Mammalian blood contains an enzymatic system, called the fibrinolytic or plasminogen system, which plays a role in various biological phenomena such as reproduction, embryogenesis, cell invasion, angiogenesis and brain function. In addition, this system participates in thrombosis, atherosclerosis, neoplasia, metastasis and chronic inflammatory disorders. The fibrinolytic system contains plasminogen, which by the action of plasminogen activators is converted to the active enzyme plasmin, which in turn digests fibrin to soluble degradation products. Two physiological plasminogen activators, respectively called tissue-type (t-PA) and urokinase-type (u-PA), have been identified. Inhibition of the fibrinolytic system may occur either at the level of plasminogen activators, by means of specific plasminogen activator inhibitors (PAI), or at the level of plasmin, mainly by means of α2-antiplasmin.
A number of substances are involved in clot formation and lysis. Plasminogen and plasmin are two of the primary substances involved in lysis. Plasminogen, a protein composed of 791 amino-acids that circulates in plasma at a concentration of about 200 μg/ml, is the zymogen form of a fibrinolytic enzyme, plasmin, which has broad substrate specificity and is ultimately responsible for degrading blood clots. For the most part, fibrin proteolysis is mediated by the generation of plasmin within a fibrin clot from the plasminogen trapped within the clot. Plasminogen-plasmin conversion, both within a clot and at its surface, is facilitated by the affinity of t-PA for fibrin, which results in a fibrin-dependent t-PA-induced plasminogen activation.
Plasminogen is a single-chain glycoprotein with a molecular weight of 92,000 which is synthesized by the liver and cleared from the circulation (via the liver) with a half-life of about 2.2 days. Human plasminogen comprises (i) a pre-activation peptide of about 67 to 76 amino-acids, (ii) five triple-loop disulfide bonded structures (named “kringles”) of about 80 amino-acids, (iii) a catalytic serine proteinase unit of about 230 amino-acids, and (iv) some inter-domain connecting sequences. Native plasminogen with NH2-terminal glutamic acid (commonly named “Glu-plasminogen”) is easily converted by limited digestion by plasmin of the Arg68-Met69, Lys77-Lys78, or Lys78-Val79 peptide bonds to modified forms commonly designated “Lys-plasminogen”. Plasminogen is converted to plasmin by cleavage of the Arg561-Val562 peptide bond. The plasmin molecule is a two-chain trypsin-like serine proteinase with an active site composed of His603, Asp646, and Ser741. The kringles of plasminogen contain lysine binding sites that interact specifically with amino-acids such as lysine, 6-aminohexanoic acid and tranexamic acid. The lysine binding sites located in the kringle 1-3 region mediate the specific binding of plasminogen to fibrin and the kinetics of the interaction of plasmin with α2-antiplasmin, and therefore play a crucial role in the regulation of physiological fibrinolysis.
Miniplasminogen is a derivative of plasminogen lacking the first four kringles which may be prepared by digestion of plasminogen with elastase and which is fully activatable to plasmin. It has a molecular weight of 38,000 and contains over 100 amino-acids of the A chain including the fifth kringle structure.
Elevated pH conditions result in cleaving the Arg530-Lys531 or Lys531-Leu532 bond of plasminogen and promoting disulfide bond rearrangement, thus producing microplasminogen, a derivative consisting of a 30 or 31 residue COOH-terminal peptide derived from the A chain bound through new disulfide bonds to the intact B-chain of plasmin, as disclosed in U.S.Pat. No. 4,774,087.
α2-antiplasmin is the main physiological plasmin inhibitor in human plasma which very rapidly inhibits plasmin, whereas plasmin formed in excess of α2-antiplasmin may be neutralized more slowly by macroglobulin and other serine proteinase inhibitors. α2-antiplasmin is a single-chain glycoprotein containing 464 amino acids which is present in plasma at a concentration of about 70 mg/l. During purification it is usually converted into a 452 amino-acid derivative by removal of 12 amino terminal amino-acids. α2-antiplasmin is synthesized by the liver and cleared from the circulation (via the liver) with a half-life of 2.6 days. Its reactive site is the Arg376-Met377 peptide bond. α2-antiplasmin is unique among serine proteinase inhibitors by having a COOH-terminal extension of 51 amino-acid residues which contains a secondary binding site that reacts with the lysine binding sites of plasminogen and plasmin. The native plasminogen-binding form of α2-antiplasmin becomes partly converted in the circulating blood to a non-plasminogen-binding, less reactive form, which lacks the 26 COOH-terminal residues. The Gln14-residue of α2-antiplasmin can crosslink to α-chains of fibrin by a process which requires Ca2+ and is catalyzed by activated coagulation factor XII. α2-antiplasmin forms an inactive 1:1 stoichiometric complex with plasmin.
Plasmin or derivatives thereof (including mini- and microplasminogen), when infused in the vicinity of a clot in a dose sufficiently high to deplete α2-antiplasmin locally in an occluded blood vessel with stagnant flow, may have a sufficiently long half-life to be able to exert a local therapeutic effect. The administration of large amounts of plasmin is well tolerated, unlike the use of certain other proteolytic enzymes.
Thromboembolic disease, i.e. blockage of a blood vessel by a blood clot, affects many adults and can be a cause of death. Most spontaneously developing vascular obstructions are due to the formation of intravascular blood clots, known as thrombi. Small fragments of a clot (emboli) may detach from the body of the clot and travel through the circulatory system to lodge in distant organs and initiate further clot formation. Heart attack, stroke, renal and pulmonary infarcts are well known consequences of thromboembolic phenomena. A blood clot is a gelled network of protein molecules within which are trapped circulating blood cells, platelets and plasma proteins. Fibrin is a major protein component of a clot which forms a relatively Insoluble network. Proteolytic, particularly fibrinolytic enzymes, have been used to dissolve vascular obstructions, since disruption of the fibrin matrix results in dissolution of the clot. Clots are formed when soluble fibrinogen, which is present in high concentrations in blood, is converted to insoluble fibrin by the action of thrombin. The probability of clot formation can be reduced by lowering the concentration of circulating fibrinogen, using fibrinogenolytic enzymes. Thromboembolytic therapies have involved the administration of a plasminogen activator, e.g. either by direct intravenous injection, or by reinjection of a patient's plasma to which a plasminogen activator has been added ex vivo, or injection of plasma protein fractions previously mixed with streptokinase, or injection of porcine plasmin stabilized with added lysine in conjunction with streptokinase.
Stroke is defined as a rapidly developing clinical sign of focal or global disturbance of cerebral function with symptoms lasting at least 24 hours. Stroke is typically caused by blockage or occlusion of blood vessels to the brain or within the brain. With complete occlusion, arrest of cerebral circulation causes cessation of neuronal electrical activity within seconds. Within a few minutes after deterioration, depletion of high energy phosphates, membrane ion pump failure, efflux of cellular potassium, influx of sodium chloride and water, and membrane depolarization occur. If the occlusion persists for more than five to ten minutes, irreversible damage results. With incomplete ischemia, however, the outcome is difficult to evaluate and depends largely on residual perfusion and the availability of oxygen. After a thrombotic occlusion of a cerebral vessel, ischemia is rarely total. Some residual perfusion usually persists in the ischemic area, depending on collateral blood flow and local perfusion pressure.
Cerebral blood flow can compensate for drops in mean arterial blood pressure from 90 to 60 mm Hg by auto-regulation. This phenomenon involves dilatation of downstream resistant vessels. Below 60 mm Hg, vasodilatation is inadequate and the cerebral blood flow falls. The brain however has perfusion reserves that can compensate for the fall in cerebral blood fall. When distal blood pressure falls below about 30 mm Hg, both compensatory mechanisms (auto-regulation and perfusion reserve) are inadequate to prevent failure of oxygen delivery. As flow drops below the ischemic threshold, symptoms of tissue hypoxia develop. Severe ischemia may be lethal. Moderate ischemia results in a tissue area that can be saved called penumbra. In the neurological context, penumbra refers to a zone of brain tissue with moderate ischemia and paralyzed neuronal function, which is reversible with restoration of adequate perfusion. The penumbra forms a zone of collaterally perfused tissue surrounding a core of severe ischemia in which an infarct has developed. When a clot is degraded and the blood flow to the penumbra is restored, the phenomenon of reperfusion injury can occur.
Although an ischemic event can occur anywhere in the vascular system, the carotid artery bifurcation and the origin of the internal carotid artery are the most frequent sites for thrombotic occlusions of cerebral blood vessels, which result in cerebral ischemia. The symptoms of reduced blood flow due to stenosis or thrombosis are similar to those caused by middle cerebral artery disease. Flow through the ophthalmic artery is often affected sufficiently to produce transient monocular blindness. Severe bilateral internal carotid artery stenosis may result in cerebral hemispheric hypoperfusion. This manifests with acute headache ipsilateral to the acutely ischemic hemisphere. Occlusions or decrease of the blood flow with resulting ischemia of one anterior cerebral artery distal to the anterior communicating artery produces motor and cortical sensory symptoms in the contralateral leg and, less often, proximal arm. Other manifestations of occlusions or underperfusion of the anterior cerebral artery include urinary incontinence due to damage to the parasagittal frontal lobe. Language disturbances manifested by decreased spontaneous speech may accompany generalized depression of psychomotor activity.
Most ischemic strokes involve portions or all of the territory of the middle cerebral artery, with emboli from the heart or extracranial carotid arteries accounting for most cases. Emboli may occlude the main stem of the middle cerebral artery, but more frequently produce distal occlusion of either the superior or the inferior branch. Occlusions of the superior branch cause weakness and sensory loss that are greatest in the face and arm. Occlusions of the posterior cerebral artery distal to its penetrating branches cause complete contra-lateral loss of vision. Difficulty in reading (dyslexia) and performing calculations (dyscalculia) may follow ischemia of the dominant posterior cerebral artery. Proximal occlusion of the posterior cerebral artery causes ischemia of the branches penetrating to calamic and limbic structures, resulting in disturbances that may chronically change to intractable pain of the defective site (thalamic pain).
A significant event in cerebral ischemia is known as the transient ischemic attack (“TIA”), defined as a neurologic deficit with a duration of less than 24 hours. TIA is an important sign of an ischemic development that may lead to cerebral infarction. Its etiology involves hemodynamic events and thromboembolic mechanisms. Because TIA often resolves within one hour, a longer deficit is often classified as presumptive stroke and is, accordingly, associated with permanent brain injury. Therefore, computed tomographic brain scans are used to search for cerebral infarction in areas affected by TIA lasting longer than two hours. Thus, the relevant clinical distinction between TIA and stroke is whether ischemia has caused brain damage, which is typically classified as infarction or ischemic necrosis. Subjects with deteriorating clinical signs might have stroke in evolution (progressive stroke).
Many other diseases are caused by or associated with ischemia. For instance, vertebrobasilar ischemia results from occlusion of the vertebral artery which causes lateral medullary syndrome with symptoms including vertigo, nausea, ipsilateral ataxia and Herner's syndrome. Vertebrobasilar ischemia often produces multifocal lesions scattered on both sides of the brain stem along a considerable length. A basilar artery occlusion produces massive deficits, including paralysis of the limbs and of most bulbar muscles, leaving the subject only able to communicate by moving the eyes or eyelids and producing an initial reduction in arousal followed by blindness and amnesia.
Venous occlusion can cause massive damage and death. The primary mechanism of brain damage is then a reduction in capillary blood flow because of increased outflow resistance from the blocked veins. Back transmission of high pressure into the capillary bed usually results in early brain swelling from oedema and hemorrhagic infarction in subcortical white matter. The most dangerous form of venous disease arises when the superior sagittal sinus is occluded. Venous occlusion occurs in association with coagulation disorders, often in the purpural period or in subjects with disseminated cancers.
Brief diffuse cerebral ischemia can cause syncope without any permanent sequel. Prolonged diffuse ischemia in other organs has devastating consequences. Common causes are cardiopulmonary failure, including infarction, aortic dissection and global hypoxia or carbon monoxide poisoning. Clinically, a diffuse hypoxia/ischemia results in unconsciousness and coma, often followed by a chronic vegetative state. If the subject does not regain consciousness within a few days, chances for the return of independent brain functions becomes very poor.
Hyperviscosity syndrome is another disease related to blood flow and ischemia. Subjects with hyperviscosity syndrome can present either with focal neurologic dysfunction or with diffuse or multifocus signs or symptoms including headache, visual disturbances, cognitive impairments or seizures.
Ischemic stroke due to thrombotic closure of a cerebral artery is amenable to therapy with antithrombotic and thrombolytic agents. The use of t-PA within three hours of symptom onset is associated with a better neurologic outcome, but a significant percentage of treated patients experience acute hemorrhage in the brain. Thus, the development of safer and more effective treatments is needed.
U.S. Pat. No. 5,288,489 discloses a method of dissolving an intravascular thrombus in a human patient, or reducing the risk of thrombus formation in a patient (such as diabetics and pregnant women), comprising administering parenterally to the patient a therapeutically effective amount of human or mammalian plasmin or mini-plasmin or micro-plasmin in a fibrinolytic or fibrinogenolytic active form, the said active form being obtained either by exposure to an insolubilized, entrapped, encapsulated or immobilized plasminogen activator or by inhibiting the autolytic activity by means of certain hydrophobic ions. This method is disclosed in the context of heart attack, stroke, renal and pulmonary infarctions, thrombophlebitis, and so on. EP-A-631,786 discloses administration to a subject of a protein having the effect of lys-plasminogen for the treatment of ischemia, infarction, brain edema and reperfusion injury that follows ischemic events. WO 00/18436 discloses the use of plasmin, mini-plasmin or micro-plasmin in a therapeutic composition for the treatment of focal cerebral ischemic infarction (ischemic stroke).
In the population over 60 years of age, the prevalence of intermittent claudication or chronic peripheral arterial occlusive disease (PAOD) being the result of atherosclerotic and thrombotic processes, is between 1 and 8%. Over the course of their disease, about 20% of the patients with intermittent claudication will progress to critical leg ischemia (acute PAOD) endangering the viability of the lower extremity, 10% will undergo invasive/surgical procedures for progressive symptoms, and 5% require amputation of the limb. Blood flow can be restored through operative bypass surgery, vascular repair surgery or pharmacological dissolution of the blood clot. Intra-arterial thrombolysis is expected to provide a significant reduction in surgical procedures, without increased risk of amputation or death. Urokinase is currently the most widely used agent for intra-arterial thrombolysis.)
Plasminogen can be obtained from human plasma fractions by affinity chromatography on lysine-Sepharose, however with yields of no more than 0.25 g/l. With the general reluctance to use plasma fractionation derivatives, alternative approaches such as production via recombinant DNA technology are preferred. For the production of a large and complex molecule such as plasminogen or plasmin, however, an effective expression system is required. Indeed recombinant Intact plasminogen cannot readily be expressed in activatable form in common eukaryotic expression systems, due to the nearly ubiquitous presence of intracellular plasminogen activators within such cell types, resulting in degradation of human plasminogen in the conditioned cell culture media. According to J. Wang et al. in Protein Science (1995) 4:1758-1767, a baculovirus/insect cell expression system has enabled expression of microplasminogen at low levels of 3 to 12 mg/l. Such a yield is obviously too low for the production of large quantities of the purified active substance. We are not aware of any data relating to the expression of miniplasminogen. Therefore, there is a need in the art for an expression system making it possible to produce large amounts of plasminogen and derivatives thereof, including mini- and microplasminogen, which will be useful in the treatment of ischemic and thrombotic disorders and associated diseases such as listed hereinabove.